1. Field of the Invention
The present invention relates to a method for γ-cyclodextrin production, and more particularly relates to a method of producing γ-cyclodextrin by using γ-cyclodextrin glycosyltransferase and isoamylase simultaneously.
2. Description of the Related Art
Cyclodextrins (CDs) are a group of structurally related natural products produced from starch or starch derivatives by catalytic action of cyclodextrin glycosyltransferase (CGTase; EC 2.4.1.19). CDs are composed of D-glucopyranose units linked by a α-1,4-glycosidic bond. The most extensively studied and exploited CDs are α-CD, β-CD and γ-CD, which are composed of 6, 7 and 8 α-1, 4 linked glucose units, respectively. The unique structure of CDs makes it easy for CDs to embed organic and inorganic compounds. Therefore, CDs are widely used in the field of food, chemistry industry, medicine, agriculture, biotechnology and textile technology. Among the three CDs, β-CD has a limited solubility in water, which allows β-CD to be easy obtained by fractional crystallization. However, the low solubility in water restricts its application as embedding compounds. Though α-CD has larger solubility in water, it is not easy to produce on industrial scale because of the low yield and costly purification. γ-CD has bigger capacity and larger solubility, which enable it to form complexes with lager molecules and improve the solubility and emulsification characteristic of the substance. At the same time, because γ-CD is safe and bland, it is possible for γ-CD to be applied in food products and medicine. Unfortunately, due to the low yield and high purification cost, the production of γ-CD can not satisfy the market needs. Only a few companies, such as Wacker and Cyclolab, produce γ-CD in a small scale. To date, γ-CD is produced from starch by amylase and γ-cyclodextrin glycosyltransferase (γ-CGTase) digestion. The starch raw material contains more than 75-85% amylopectin, which means there is one α-1,6-glycosidic bond every 17-28 glucose units. Because the γ-CGTase can not hydrolyze the α-1,6-glycosidic bond, the γ-CD yield is very low and the production cycle is very long.
Isoamylase (EC 3.2.1.68) which is mainly used for food additives is a hydrolase that catalyzes the hydrolysis of α-1,6-glycosidic branch linkages in glycogen and amylopectin. During CDs production, isoamylase can be added to hydrolyze the α-1,6-linkages, and then, the CGTase carry out the cyclization reaction. Therefore, methods of using isoamylase and CGTase were developed to improve the yield and lower the cost of CDs production. Ivan Pishtiyski (Pishtiyski, I. and B. Zhekova (2006). “Effect of different substrates and their preliminary treatment on cyclodextrin production.” World Journal of Microbiology and Biotechnology 22(2): 109-114) achieved 65% yield of CDs using 2.5% potato starch as substrate and a two-step conversion reaction using pullulanase for debranching and CGTase for cyclization. Rendleman (Rendleman, J. A. Jr. (1997). “Enhancement of cyclodextrin production through use of debranching enzymes.” Biotechnology and applied biochemistry 26(1): 51-61.) achieved 76% yield with 10% waxy corn starch as substrate by using debranching enzyme (pullulanase and isoamylase) and α-CGTase for cyclization at 15° C. However, the concentration of the substrate was too low and the reaction time took as long as 5 days. Isoamylase is almost inactivated under alkalic condition and wild-type γ-CGTase only has 10% relative activity under neutral pH. As a result, there is no report of using isoamylase and γ-CGTase simultaneously for γ-CD production. The relative activity of a γ-CGTase mutant, γ-CGTase (A223K) from Alkalophilic Bacillus 7364 (Nakagawa Y, Takada M, Ogawa K, Hatada Y, Horikoshi K. “Site-directed mutations in Alanine 223 and Glycine 255 in the acceptor site of gamma-Cyclodextrin glucanotransferase from Alkalophilic Bacillus clarkii 7364 affect cyclodextrin production.” J. Biochem. 2006 140(3):329-36.), has more than 3-fold increase under neutral pH (pH 7.5) when compared to that of the wild type enzyme, which makes it possible to use isoamylase and γ-CGTase (A223K) simultaneously for γ-CD production. The present invention provides a method for producing γ-CD in one-step conversion reaction using isoamylase and γ-CGTase (A223K) under a neutral pH.